Dr. Hem Shah,S15729, Dr. Prajna N V, Dr. Hemadevi Boomiraj,
Dr. Prajna Lalitha Dr.Hem Shah, Dr. Lalitha Prajna, Dr. N.V.Prajna.
Background:
Microbial keratitis due to either fungus or bacteria is a major cause of blindness in India. Majority of times inspite of adequate medical management the ulcer does not heal and may require a corneal transplant. One of the major reasons for a poor outcome in these diseases is the excessive inflammation that is mounted by the host to overcome these infections.
MicroRNAs (miRNAs) are small, stable non-coding RNA molecules with regulatory function and marked tissue specificity that post-transcriptionally regulate gene expression. MiRNAs are expressed in various ocular tissues and have been implicated in human corneal diseases like keratoconus, Fuchs’ dystrophy and Herpetic stromal keratitis. Our previous study on the human miRNA expression profile in fungal keratitis corneas showed dysregulated miRNA expression that could be associated with excessive corneal inflammation and impaired wound healing in fungal keratitis. In this study, expression of selected dysregulated miRNAs in fungal keratitis was compared to non-infectious corneal inflammatory disease.
Methodology:
Samples: Post-transplant corneal buttons (n=5) from Aspergillus flavus keratitis and pseudophakic bullous keratopathy (n=3) were obtained. Human donor eyes were acquired from Rotary Aravind International Eye Bank (Madurai, India) and the corneas were trephined immediately. Corneal tissues were snap frozen in liquid nitrogen and stored at -80°C.
Real time quantitative polymerase chain reaction: Total RNA was extracted with Trizol method following manufacturer’s protocols. Mature microRNAs were polyadenylated and reverse transcribed using miScript II RT Kit (Qiagen, Valencia, CA) whereby a universal tag is incorporated at the 5’end of the cDNA. This tag is bound by the universal reverse primer during real time PCR amplification with miScript SYBRTM Green PCR Kit (Qiagen, Valencia, CA), and custom synthesized forward primers. The reaction conditions included an initial activation step at 95°C for 15 min, followed by 40 cycles of 94°C for 15s, 55°C for 30s and 70°C for 40s. Data were normalized using RNU6B (U6) snRNA levels. For quantitative analysis of mRNA expression, cDNA was synthesized using high capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA) and qPCR was done with power SYBR green PCR master mix (Applied Biosystems, Carlsbad, CA) and custom made primers. PCR amplification was carried out at 50°C for 2min, 95°C for 10min and 40 cycles of 95°C for 15s, 60°C for 1min with a final melt curve analysis. The reactions were run in triplicates and the results were normalized using β-actin mRNA levels. The relative expression between control and patient samples was calculated by 2^-DDCt method.
Results:
Earlier study was based miRNA expression profile in human cornea from fungal keratitis patients infected with Aspergillus flavus. By Illumina deep sequencing, a total of 75 miRNAs were significantly differentially expressed in pooled keratitis corneas compared to normal. In continuation, to identify their role on corneal inflammatory disease, hsa-miR-223-3p, hsa-miR-142-3p, hsa-miR-204-5p, hsa-miR-511-5p, hsa-miR-451a and novel mir-cornea-3p were selected. Corneas from pseudophakic bullous keratopathy patients were used to represent non infectious inflammatory disease. Relative expression of miRNA was analysed by real time qPCR using cadaver corneas as controls.
Notably, expression of miR-223-3p was several fold higher in fungal keratitis corneas compared to bullous keratopathy, and it’s target NLRP3 was negatively expressed which further indicates the infiltrating neutrophils as a major source of this miRNA during corneal infections. Whereas, the dysregulated miR-204-5p in fungal keratitis was upregulated in bullous keratopathy.
Conclusion:
Study highlighted the role of miR-223-3p during infectious corneal inflammation and miRNA 451-a in corneal inflammation irrespective of infection. This work indicates that miRNAs play important regulatory roles in corneal inflammation and further investigation is required for selected miRNAs as candidates for clinical use.